We found that patatin and Kunitz protease inhibitor KPI variants are extraordinarily dominant in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras.
Trang 1proteins in tuber of potato cv Kuras
Guy Bauw, Heidi V Nielsen, Jeppe Emmersen, Ka˚re L Nielsen, Malene Jørgensen and
Karen G Welinder
Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark
Potato (Solanum tuberosum) is the world’s fourth most
important crop after rice, wheat and corn Originating
in South America, this plant is now cultivated and
consumed worldwide The potato tuber is also an
industrially important source of starch Centuries of
breeding have resulted in thousands of variants
adap-ted to specific climatic, agricultural, pest resistance,
nutritional, sensory and industrial requirements
Despite the importance of the potato tuber for the
food and starch industries, detailed knowledge of
potato tuber proteins is scarce Potato tuber proteins
are classified into three groups: patatins, protease
inhibitors, and other proteins [1] The patatin and pro-tease inhibitor classes, which form the bulk of the potato tuber protein, are mostly considered to be stor-age proteins The majority of the isoforms have defined enzymatic and inhibitory activities that might
be of physiological relevance For example, several studies have indicated that purified patatin and certain protease inhibitors reduce the growth of larvae [2–5] Potato tuber protease inhibitors are a diverse group
of proteins that inhibit a variety of proteases and some other enzymes, for example invertase [6] Some have dual or broad substrate specificity [4] They differ in
Keywords
Kunitz protease inhibitor; patatin;
Solanum tuberosum; lipoxygenase; cultivar
markers
Correspondence
K G Welinder, Department of
Biotechnology, Chemistry and
Environmental Engineering, Aalborg
University, Sohngaardsholmsvej 49,
DK-9000 Aalborg, Denmark
Tel: +45 963 58467
E-mail: welinder@bio.aau.dk
(Received 5 April 2006, revised 18 May
2006, accepted 7 June 2006)
doi:10.1111/j.1742-4658.2006.05364.x
The major potato tuber proteins of the Kuras cultivar, which is the domin-ant cultivar used in Northern Europe for industrial starch production, were analysed using 1D and 2D gel electrophoresis The electrophoretic patterns varied significantly depending on the method of preparation and the potato variant (Solanum tuberosum) Proteins were characterized using MS and scored against potato protein databases, derived from both ‘Kuras only’ and ‘all potato’ expressed sequence tags (EST) and full-length cDNAs Despite the existence of 180 000 ESTs, the currently available potato sequence data showed a severe under-representation of genes or long tran-scripts encoding proteins > 50 kDa (3.5% of all) compared with the com-plete proteome of Arabidopsis thaliana (33% of all) We found that patatin and Kunitz protease inhibitor (KPI) variants are extraordinarily dominant
in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras Other proteins identified include annexin, glyoxalase I, enolase and two lipoxygenases, the sequences of which are highly conserved among potato variants Known S tuberosum patatins cluster into three clades all represented in Kuras S tuberosum KPIs cluster into more diverse clades of which five were found in Kuras tuber, including a novel clade, KPI K, found to date only in Kuras Furthermore, protein abun-dance was contrasted with the levels of corresponding gene transcripts found in our previous EST and LongSAGE studies of Kuras tuber
Abbreviations
EST, expressed sequence tag; IPG, immobilized pH gradient; KPI, Kunitz protease inhibitor; pI, isoelectric point; PMF, peptide mass fingerprint; SAGE, serial analysis of gene expression; St, Solanum tuberosum.
Trang 2their amino acid sequence, chain length (Mr 4, 8, and
20–22 kDa) and subunit composition (monomer to
pentamer) [7] According to the recently introduced
general classification of protease inhibitors [8], potato
tuber contains inhibitors of five nonhomologous
super-families I3A, I13, I20, I25B and I37 (http://www
merops.ac.uk)
By contrast to potato protease inhibitors, patatins
constitute a more uniform group of homologous
pro-teins Pots et al [9] separated the patatins of the Bintje
cultivar into four pools with different chromatographic
and electrophoretic characteristics, but similar
biophys-ical properties Patatins catalyse the nonspecific
hydro-lysis of a wide range of acyl and phospholipids [5,10]
Genes encoding patatins [11,12], and Kunitz protease
inhibitors (KPIs) [13–15] of different potato cultivars
have been cloned and sequenced Based on genetic and
molecular analysis, Twell and Ooms [12] estimated that
there are 64–72 patatin DNA copies in the tetraploid
genome of potato cv Desiree
Little information on other proteins present in the
tuber is available, and until recently, no systematic
gene discovery or protein sequencing had been
under-taken Only a few studies have reported on the protein
content and its regulation upon physiological changes
The protein content of the potato tuber functioning as
a sink during maturation has been compared with the
tuber acting as a source for sprouting and the
initi-ation of a new plant by Borgmann et al [16] Espen
et al [17] reported on protein changes upon tuber
dor-mancy, whereas De´sire´ et al [18,19] used agar-grown
microtubers to analyse protein changes due to
tuberi-zation and the breaking of dormancy None of these
reports analysed the proteins of interest by sequencing
Recently, Lehesranta et al [20] identified different
pro-teins from the cultivar Desiree using LC-MS⁄ MS, as
part of an extensive comparison of the 2D gel
elec-trophoretic protein profiles of different potato variants,
and the influence of genetic engineering on the potato
tuber protein profile
Potato gene discovery using expressed sequence tag
(EST) sequencing [21–23] (http://www.tigr.org) has
recently generated a large number of partial nucleotide
sequences, originating from different potato cultivars
and tissues These partial sequences, together with the
limited number of full-length cDNA sequences have
been assembled in continuous nucleotide sequences or
contigs, generating a plethora of potential protein
sequences
The Kuras cultivar, having a superior pest
resist-ance, is the major starch potato grown in Northern
Europe A large number of EST sequences expressed
in the mature tuber are available for this particular
cultivar [21], together with an increasing number of full-length cDNA sequences (HV Nielsen, KL Nielsen
& J Emmersen, unpublished results) Furthermore, the transcriptome of the mature tuber cv Kuras was recently analysed using serial analysis of gene expres-sion (LongSAGE), which generated 19 nucleotide sequence tags of the expressed genes [24] Here we report on electrophoretic separations of Kuras tuber proteins, the protein chemical characterization of the major proteins and the classification of currently known patatins and KPIs into clades based onto sequence similarity This study provides clear evidence that the protein variants within the three distinct clades
of patatins pat1, pat2 and pat3, and the five clades of Kunitz protease inhibitors, KPI A, KPI B, KPI C, KPI K (Kuras), KPI M (miraculin-like) expressed in Kuras tuber, are cultivar specific Moreover, to date, KPI K has been found only in cv Kuras In contrast, the amino acid sequences of other major proteins such
as annexin, glyoxalase I, enolase, and lipoxygenase showed little, if any, sequence variation among potato variants
Results
Electrophoretic patterns of potato tuber proteins from cv Kuras
2D-PAGE Protein was extracted from mature field-grown potato tubers (S tuberosum cv Kuras) and separated using 2D-PAGE, using a broad range immobilized pH gradi-ent (IPG) pH 3–10 as the first dimension (Fig 1) Vis-ual inspection of the protein patterns of individVis-ual tubers did not reveal major differences The number of individual protein spots detectable is limited as two protein groups make up the majority of tuber proteins
in Kuras, the overlapping patatins with a molecular mass of 40–45 kDa, isoelectric point (pI) 4–5, and the KPI with a molecular mass of 20–22 kDa spread over the entire pI range
To improve the resolution, proteins were separated
on IPG strips, pI 4–7 and 6–11 On a typical 2D gel,
pI 4–7, developed using Coomassie Brilliant Blue, epi-cocconone or silver staining, 250, 550 and 600 protein spots were detected, respectively (supplementary Fig S1)
In general, a four- to eightfold higher amount of protein was applied to 2D gels stained with Coomassie Brilliant Blue and used for protein analyses (Fig 2) Apart from the major proteins at 20–22 and 40–
45 kDa, few proteins are seen in Coomassie Brilliant Blue-stained gels, even after enhancement with computer software Staining with the more sensitive
Trang 3epicocconone or silver stains revealed more spots
dis-tributed throughout the pI and Mrranges
On a Coomassie Brilliant Blue-stained gel (pI 6–11)
only a few well-defined protein spots at 20–22 kDa
were visible Epicocconone or silver staining revealed
300 and 280 protein spots, respectively, most on the
neutral side of the 2D gel (supplementary Fig S1)
The intensity and the area of the minor protein spots
were at least two orders of magnitude lower than the
abundant proteins visible using Coomassie Brilliant
Blue staining (Fig 2)
Unmodified versus reduced and alkylated 2D-PAGE
protein patterns
Figures 1 and 2 show unmodified tuber protein (i.e
without reduction and alkylation at any stage of the
preparation), which we found most reproducible, and
which retained disulfide-linked subunits in one protein
spot Reduction and alkylation, which have been used
in most studies, resulted in altered 1D and 2D patterns
(Fig 3, supplementary Fig S2) The most remarkable
changes were observed for the 20–22 kDa KPIs
Redu-cing the IPG-focused protein by including 50 mm
dithiotreitol in the SDS equilibration buffer before 2D
electrophoresis, changed the position of some protein
spots This indicates a more complete unfolding due to
disulfide bond cleavage (decreased mobility), or
clea-vage of disulfide-linked subunits (increased mobility)
When the reduction was performed before any
elec-trophoretic separation, both pI and size changed for the same reasons Additional alkylation of cyste-ines using the neutral iodoacetamide (supplementary Fig S2D) should look similar to reduction only (sup-plementary Fig S2C), but showed less-distinct spots for unknown reasons Alkylation with iodoacetic acid introduces negative charges at all cysteines, resulting in lower pI values This shifted almost all 20–22 kDa pro-teins to the pI 4–7 region (supplementary Fig S2E)
Tuber proteins of molecular masses 50–120 kDa Protein spots with higher molecular masses varied in intensity from 2D gel to 2D gel, probably due to restricted entry into the polyacrylamide network Fur-thermore, because many of these proteins are resolved
in spot series of identical mass, the tuber proteins were separated by 1D SDS⁄ PAGE using a cross-linking of 0.5% instead of 2.6% to improve protein yield, separ-ation and reproducibility (Fig 3) The intensities of the individual protein bands in the SDS gel provided
an estimate of their relative abundance The mobility
of proteins changed upon reduction of disulfide bridges
as seen by comparing lanes 2 and 3 of Fig 3
Identification of potato tuber proteins Thirty-nine unmodified protein spots from Coomassie Brilliant Blue-stained 2D gels (Fig 2) were assigned using tryptic peptide mass fingerprint (PMF) analysis
Mr
66 45 35
25
18
(kDa)
Fig 1 Broad pI range 2D gel of potato (cv.
Kuras) tuber proteins Protein was extracted
from potato tuber and separated by
isoelec-tric focusing IPG pH 3–10 in the first
dimen-sion and by 12.5% SDS ⁄ PAGE in the
second dimension One milligram of
unmodified protein (i.e not reduced or
modified) was applied, and the proteins
stained using epicocconone.
Trang 4and curated manually (Table 1) Fifteen reduced and
alkylated potato tuber protein spots were also
identi-fied by PMF (data not shown), and confirmed the data
given in Table 1
S tuberosum patatins
The prominent acidic protein spots of 40–45 kDa were
patatin variants (Table 1, Fig 4) Kuras EST patatin
sequences were assembled into eight contigs and two
singletons [25] (http://www.bio.aau.dk/en/st-data.htm),
and were confirmed using full-length sequencing of
seven different clones (GenBank accession numbers DQ114415–DQ114421) The phylogenetic tree based
on the patatin cDNA sequences (Fig 5) showed that the Kuras patatin genes cluster in three clades, pat1 with the Kuras-specific pat1-k1, -k2 variants, pat2 which is more diverse including Kuras-specific pat2-k1, -k2, -k3, -k4 variants, and pat3 with only a single form
in Kuras, pat3-k1 The patatin sequences showed pronounced cultivar-dependent variation among the currently known potato patatin genes (Fig 5) and proteins (Fig S3) Kuras patatins are 84–96% identical
in terms of their amino acid sequences, and have 80– 95% identity with published potato patatin sequences from various strains
The higher mass patatin spots 27–31 (Fig 2) were identified as pat3-k1, which contains three potential N-glycosylation sites, two of which were confirmed using PMF analysis (Fig 4) This prominent patatin
Mr
(kDa)
66
45
35
25
18
25
3
A
B
C
7 pI
18
Fig 2 Annotation of the protein spots on Coomassie Brilliant
Blue-stained gels (A) 2D gel pI 4–7 of potato tuber proteins (B)
Enlarge-ment of patatin spots boxed in gel (A) (C) Lower half of the 2D gel
pI 6–11 of potato tuber.
Mr
SP1 Lip
Eno
Pat
200 150 120 100 85
70 60 50
40
30
Fig 3 SDS ⁄ PAGE separation of high molecular mass potato tuber proteins stained with Coomassie Brilliant Blue Lane 1, molecular mass markers; lane 2, protein reduced with dithiotreitol and alkylated with iodoacetic acid; lane 3, unmodified protein extract Predomin-ant identified proteins of lane 3 are indicated by arrows, starch phosphorylase 1 (SP1), lipoxygenase (Lip), enolase (Eno) and pata-tin (Pat).
Trang 5Table 1 Identification of 2D gel-separated tuber proteins from mature field-grown potato (cv Kuras).
Spot Protein Accession number Matching peptides
Sequence a
coverage (%)
Experimental Calculated a
pIb M r (kDa) pI M r (kDa) Acidic proteins pI 4–7:
18 Patatin pat1-k2,
pat2-k1
DQ114416,e DQ114417 e
20 Patatin pat1-k2,
pat2-k1 ⁄ k3
21 Patatin pat1-k2,
pat2-k1 ⁄ k3
24 Patatin pat1-k2,
pat2-k2
Basic proteins pI 6–11:
a Mature protein (removal of signal sequence and known pro-peptides) b pI predicted with cystine (SS) using GPMAW [46] c Tentative contig number with best match Kuras proteins deviate at certain positions. d TIGR accession numbers at http://www.tigr.org/tigr-scripts/tgi/ T_index.cgi?species=potato Tentative contigs might be > 95% identical to Kuras-specific sequence e GenBank accession of full-length cDNA sequence of Kuras (HV Nielsen, KL Nielsen & J Emmersen, unpublished results) f Kuras contigs accession number at http:// www.bio.aau.dk/en/st-data.htm.
Trang 6has most likely lost its lipolytic activity due to a Ser to
Gly substitution (position 54) (Fig 4) in the proposed
catalytic centre [26], which supports its role as a
stor-age protein
Protein spots 15 and 16, pat2-k3, and 17 and 19,
pat1-k1, seemed to be single patatins (Table 1) The
MS spectra of the remaining patatin spots 18 and
20–26, contained tryptic peptide mass values derived
from different Kuras patatin variants, as they included
masses of homologous peptides from two variants (e.g
spot 18 contained pat1-k2 and pat2-k1, Fig 4) None
of the potential N-glycosylation site(s) of the pat1 and
pat2 variants were verified by tryptic peptides
Although pat1-k1 contains two potential
N-glycosyla-tion sites, one in common with pat3-k3, the remaining
patatins have only a single potential site All isopatatin
spots 15–31 showed a mass peak of 1705.9 ± 0.2 Da
corresponding to their identical N-terminal peptides,
TLGEMVTVLSIDGGGIK (Table 2) The unique
N-terminal peptide of pat1-k2 was not seen in spot 18 (Table 2) An overview of the patatin results are shown
in Fig 4
S tuberosum Kunitz protease inhibitors (StKPI) The prominent 20–22 kDa proteins were distributed over the entire pI range belong to the KPI family, which includes the classical soybean trypsin inhibitor Our results are best presented in the context of a con-sistent nomenclature based on sequence similarity The protein sequences of KPIs in various plants, including Arabidopsis and tomato, contain the 17-residues KPI signature [LIVM]-x-D-{EK}-[EDHNTY]-[DG]-[RKHDENQ]-x-[LIVM]-x-{E}-x-x-x-Y-x-[LIVM], where H is observed in some potato miraculin-like seq-uences ([], residues allowed, and {}, residues excluded from the position; X, any residue) (http://www.expasy org/cgi-bin/nicedoc.pl?PDOC00255) [27] In Kuras tubers
Fig 4 Alignment of amino acid sequences of the mature patatin proteins of potato cv Kuras using pat1-k1 as template (.) Identical to tem-plate; (–) gap The single Cys162 conserved in all patatins, and the catalytic Ser54 substituted to Gly in pat3-k1 are shown in bold Potential N-glycosylation sites are shown in small italics or bold capitals, if verified by PMF The predominant potato protein N-linked glycan [48] (Xyl)Man3(Fuc)(GlcNAc)2gives rise to a mass increase of 1171 Da Grey shaded residues were covered by peptide masses of the PMF ana-lysis of a particular 2D spot, pat1-k1 ¼ spot 19, pat2-k3 ¼ spot 16, pat3-k1 ¼ spot 28 Both pat1-k2 and pat2-k1 were present in spot 18 Underlined sequences were identified by nanospray MS ⁄ MS analysis Corresponding GenBank accession numbers are found in Table 1.
Trang 7we found five StKPI clades represented (Figs 6,7;
sup-plementary Fig S4): KPI A, KPI B, KPI C, in
accord-ance with the nomenclature of Ishikawa et al [14]
(cv Danshaku) and Heibges et al [15] (cv Provita and
cv Saturna); KPI K (to date found only in Kuras);
and KPI M, similar to the sweet tasting miraculin
des-cribed by Theerasilp et al [28]
Thirteen Kuras KPI sequences of the KPI A, B,
and C clades were subjected to full-length cDNA
sequencing (GenBank accession numbers DQ168311,
DQ168316–DQ168319, DQ168324, DQ168325,
DQ168327, and DQ168329–DQ168333) Some of these
sequences differ only outside the reading frame
Ku-ras-specific contigs and tentative contigs, for all known
potato ESTs are available at http://www.bio.aau.dk/
en/st-data.htm and http://www.tigr.org, respectively
Full-length cDNA sequences, a contig for KPI K
(K2-01900, TC112888), and two contigs for KPI M
(K1-01724, TC112274 and TC112554) may account for the
tryptic peptide data of protein spots 1–7, 9–11, and
61–69 (Table 1, Fig 6) Masses verifying the N- and
C-termini of mature KPIs were observed for variants
of the KPI A, KPI B and KPI C clades (Table 2, Fig 6)
The 2D gels show that KPI C variants all have basic
pI values of 7.0–8.6, whereas KPI A, KPI B, KPI K and KPI M variants are acidic to neutral with experi-mental pI values of 4.0–6.0 (Fig 2)
Analysis of StKPI sequences showed pronounced cultivar-dependent variation among the known genes (Fig 7) and proteins (Fig S4) The amino acid identity among variants within each of the five clades is
> 83% for Kuras extending to 77% including all culti-vars, whereas the StKPI interclade identity varies from
75 to 15% Therefore, StKPIs constitute a much more diverse protein family than St patatins
Other major proteins
In addition to the dominating patatins and KPIs in Kuras, five other proteins, annexin, glyoxalase I, eno-lase, and two of unknown function were identified from the 2D-gel (Table 1; Fig S5) Masses correspond-ing to acetylated N-termini of mature annexin and gly-oxalase I, together with their C-termini were observed
in the MS spectra (Table 2)
The 50–120 kDa region of reduced tuber protein separated by 1D SDS⁄ PAGE (Fig 3, lane 2) was cut into successive 10· 2 mm slices from an unstained part of the gel, digested with trypsin and analysed, whereas discrete protein bands were cut from unmodi-fied protein (Fig 3, lane 3) The mass spectra of the 1D samples show a higher background level of low-intensity ions from minor proteins as expected There-fore, a total of 70–120 intense and well-defined mass values were collected from the nine mass spectra of each gel band Fourteen different proteins were identi-fied unambiguously by at least 15 tryptic peptide masses, most in 2–4 adjacent gel slices (Table 3; sup-plementary Table S1)
In contrast to the patatin and KPI protein families, our extensive analysis of the amino acid sequences of other major proteins, i.e glyoxalase I, annexin, eno-lase, cataeno-lase, UTP : glucose 1-phosphate uridyltrans-ferase (UDP pyrophosphorylase) (supplementary Fig S5), revealed no or only very limited (< 2%) sequence variation among S tuberosum strains
Discussion
Tuber proteins from mature field grown Kuras pota-toes were characterized using 1D and 2D gel electro-phoresis, and the major proteins identified by peptide mass fingerprinting Unmodified protein provided the most reproducible 2D pattern We show how the gel
Fig 5 Phylogenetic tree of S tuberosum patatin cDNA
sequences The tree was constructed using minimum evolution
dis-tance analysis The corresponding protein sequences and
refer-ences are shown in supplementary Fig S3 Bootstrap values of
1000 resamplings are indicated at the nodes of the tree Non-Kuras
patatins genes are indicated with their GenBank accession
num-bers Scale bar indicates five substitutions per 1000 nucleotides.
Trang 8electrophoretic patterns of potato tuber proteins
change with common chemical treatments such as
disulfide reduction and alkylation The rather high
sequence coverage obtained in this study was essential
to the successful distinction among the many cultivar
specific patatin and KPI variants Separation of the
tryptic peptide mixtures using stepwise elution from
Poros beads combined with the use of different
MALDI matrices [29,30] provided this high coverage
For some proteins, the maximum possible sequence
coverage was reached, because missing sequences were
either small hydrophilic tryptic peptides (fewer than
seven residues) or long hydrophobic ones with low
recoveries from Poros beads and masses outside the
MALDI-reflectron TOF window Nanospray MS⁄ MS
analyses provided only limited additional information
(Figs 4, 6) N- and C-terminal tryptic peptides of
mature proteins were accounted for in several cases
(Table 2)
Patatins
Seven patatins have been cDNA sequenced and also
identified as proteins in mature Kuras tuber All
known S tuberosum patatins are distributed into
three clades based on sequence similarity, and those
of Kuras are cultivar dependent Also the relative
abundance of patatin in tubers shows a marked
dependence on cultivar [1,31,32] The statistical analy-sis of the abundance of 2D gel separated proteins of
> 20 potato cultivars, landraces and genetically modi-fied potatoes by Lehesranta et al [20] demonstrated a high variability of patatins in general Comparing the similarly prepared tuber protein and 2D gels of Kuras and of Desiree, Maris Piper and a landrace of Lehes-ranta et al [20], the patatin content in Kuras is remarkably high Comparing total protein distribution
of some common Danish potato varieties by SDS⁄ PAGE supported this greater abundance of patatins in Kuras (M Jørgensen & K.G Welinder, unpublished results)
Kunitz protease inhibitors Protein reduction changed the pattern of KPIs signifi-cantly, due to the cleavage of two conserved disulfide bonds [33], at positions Cys88–Cys141 and Cys197– Cys214 (Fig 6) In fact, KPI clades show an extensive variability within the last cystine loop KPI A has an insertion, which is present at both the nucleotide and protein levels KPI B has a variable insertion at the nucleotide level When present, it is removed as a pro-peptide at the protein level This gives rise to separ-ation into a larger N-terminal and a smaller C-terminal subunit of KPI B after reduction (supple-mentary Fig S2) The two-chain structure of this type
Table 2 Masses of the N- and C-terminal tryptic peptides of 2D gel proteins All experimental masses deviated < ± 0.2 Da from the calcu-lated monoisotopic masses.
Calculated mass
N-terminal peptides:
C-terminal peptides:
a ac, acetyl (+ 42.0 Da) b Met detected as methionine sulfoxide.
Trang 9of KPI was first documented by Valueva et al [33]
using complete amino acid sequencing KPI C and
KPI M (miraculin) have no inserts at the nucleotide
level, and produce continuous peptide chains on
reduc-tion, similar to KPI A KPI K has the nucleotide
insert The recognition of a peptide ion mass of
2197.15 covering the sequence
LLGYELITCD-GALVTMGQR (Cys-acrylamide, methionine
sulfox-ide) indicates that this insert might be retained in the mature protein, although unambiguous verification is required KPI K is unique to Kuras so far and was represented by 24 ESTs (TC112888; K2-01900) [21] It will need further structural and functional characteri-zation Contrasting with KPIs, patatins changed only
on carboxymethylation due to the presence of a single cysteine only (Cys162) (Fig 4)
Fig 6 Alignment of Kunitz protease inhibitors identified in Kuras potato tubers (.) Identical to template residue of the KPI clade; (–) gap; (italics) ER signal sequence predicted by SIGNALP [49] Cystein residues are shown in bold Cys88–Cys141 and Cys197–Cys214 are expected
to form disulfide bridges [33] Grey shaded residues were covered by the masses of the PMF analysis of a particular 2D spot, KPI A-k1 ¼ spot 9, KPI B-k1 ¼ spot 4, KPI B-k2 ¼ spot 6, KPI B-k3 ¼ spot 10, KPI B-k4 ¼ spot 6, KPI C-k1 ¼ spot 63, KPI C-k2 ¼ spot 68, KPI C-k3 ¼ spot 69, KPI K-k1 ¼ spot 11, KPI M1 ¼ spot 1, KPI M2 ¼ spot 5 Underlined sequences were identified by nanospray MS ⁄ MS analysis Corresponding GenBank accessions are found in Table 1 The 17 residues Kunitz motif is boxed by a solid line The highly variable, absent or split sequence in KPIs is boxed by a dotted line.
Trang 10Potato protein databases
The identification of potato tuber proteins of 50–
120 kDa was limited due to their variable intensities in
2D gels, and due to the present severe
under-represen-tation of long cDNAs and contigs among 180 000
ESTs Thus the assembled TIGR tentative contigs
cod-ing for potato proteins with more than 450 amino acid
residues constituted only 3.5% (1314 sequences) of all,
whereas this is 33% (± 12 000 sequences) for the
complete Arabidopsis thaliana proteome Also potato
proteins 30–50 kDa predicted from EST contigs
accounting for 12% (4465 sequences) of all were
under-represented compared with the size distribution
within the A thaliana proteome (33%) To reduce the
occurrence of false positives, identification of 1D gel separated proteins was presently restricted to those with 15 or more matching masses, sequence coverage
of 25% or more, and identification in at least two adjacent gel slices (supplementary Table S1) A further limitation of this study was the absence of small pro-teins 3–10 kDa in the protein gels, such as carboxy-peptidase inhibitors, which can be obtained by chromatography [7]
Potato tuber proteome versus transcriptome The protein ensemble of tuber is of interest to the potato starch industry, as predominant proteins might
be purified in quantity from potato juice, a waste from
Fig 7 Phylogenetic tree of S tuberosum KPI cDNA sequences The tree was con-structed similarly to Fig 5 The correspond-ing protein sequences and references are shown in supplementary Fig S3.